Biochemistry, Molecular Biology, Physiology, Microbiology, Immunology, Pharmacology & Drug Discovery
Preparation Lecture Notes, Multiple Choice Questions, Scientific Discoveries

Medical Microbiology: Chlamydia trachomatis and related micorganism MCQ

February 27, 2019
Multiple Choice Question with answers on Chlamydia trachomatis & other Chlamydiaceae

1) Which of the following Chlamydia spp are human pathogens? Select from all the options given below
a) Chlamydia pecorum
b) Chlamydia psittaci
c) Chlamydia trachomatis
d) Chlamydia pnuemoniae

2) What serotypes of Chlamydia trachomatis is associated with inclusion conjunctivitis seen in adults and neonates?
a) Serotypes D to K
b) Serotypes L1,2 and 3
c) Serotypes A, B, and C
d) Serotypes A, B, B and C

3) What type of laboratory methods are used for the diagnosis of genital Chlamydia trachomatis infections? Select all that applies
a) Specimen culture in McCoy cell tissue culture
b) Direct fluorescent antibody and Enzyme-linked immunoassay
c) Serological tests
d) Nucleic acid amplification test


4) Chlamydia trachomatis is transmitted from infected mother to baby through the birth canal and can cause neonatal inclusion conjunctivitis, how many days after the baby is born symptoms in eyes start to develop?
a) After 24 hours
b) Within 48 hours
c) After 6 hours
d) Within 7 to 15 days

5) All of the following statements are true about the antigens of Chlamydia spp, Except?
a) Genus-specific antigens are common to all Chlamydia
b) Only Chlamydia trachomatis have genus-specific antigens
c) Species-specific antigens are present in the outer membrane of Chlamydia spp
d) None of the above

6) Which of the following statements is NOT the distinguishing features of the family Chlamydiaceae?
a) They are seen on Gram stain
b) They are obligate intracellular bacteria
c) They cannot make ATP
d) They consist of two forms elementary and reticulate body

7) All of the statements given below are correct about Chlamydia psittaci, EXCEPT?
a) It is a primary pathogen of birds
b) It can cause respiratory infections in human
c) It is usually resistant to sulfonamides
d) It only infects birds like parrots and cockatoos

8) What is the most sensitive laboratory diagnostic tests for Chlamydia pneumoniae?
a) Giemsa staining test
b) Microimmunofluorescence test
c) Cell culture method
d) None of the above

9) Human is the only reservoir of Chlamydophila pneumoniae, the pathogen is most prevalent in which age group of people?
a) 2 to 5 years
b) 10 to 30 years
c) 5 to 18 years
d) 20 to 45 years


10) Name the organism given below which is frequently associated with urethritis apart from Chlamydia trachomatis?
a) Klebsiella pneumoniae
b) E. Coli
c) Streptococcus agalactiae
d) Neisseria gonorrohoea

11) All of the statements given below are correct about pathogenic Chlamydophila pneumoniae, EXCEPT?
a) It causes respiratory infections which is most common in young children
b) The bacteria spread from person to person via a respiratory route
c) It produces intracytoplasmic inclusions that lack glycogen
d) The infections caused by the bacteria are usually asymptomatic

12) When do the symptoms of neonatal pneumoniae in babies start to show caused by Chlamydia trachomatis?
a) In 1 week after the birth
b) In 2 to 12 weeks after birth
c) In 5 to 10 days after the birth
d) In 2 to 3 weeks after the birth

Multiple Choice Answers Review
1- Option b, c, and d are correct
2-a) Serotypes D to K
3-Option a, b, c & d
4- d) Within 7 to 15 days
5-b) Only Chlamydia trachomatis have specific antigens
6-a) They are seen on Gram stain
7-d) It only infects birds like parrots and cockatoos
8-b) Microimmunoflourescence test
9-c) 5 to 18 years
10-c) Neisseria gonorrohoea
11-a) It causes respiratory infections which is most common in young children
12-b) In 2 to 12 weeks after birth
Medical Microbiology: Chlamydia trachomatis and related micorganism MCQ Medical Microbiology: Chlamydia trachomatis and related micorganism MCQ Reviewed by Biotechnology on February 27, 2019 Rating: 5

AVV5 mediated microRNA delivery for treatment of ALS

February 25, 2019


AAV5 mediated delivery of micro-RNA for treatment of ALS and FTD with C9orf72 G4C2 expansion

Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons, leading to muscle atrophy and paralysis. A significant number of ALS patients also develop frontal temporal dementia (FTD) caused by progressive degeneration of frontal and temporal lobes. There are no disease-modifying drug and most patients die within 3-5 years after the onset of the disease. The most common genetic cause of ALS and FTD is an expansion of G4C2 repeats (> one hundred repeats) in the first intronic site of the c9orf72 gene present on chromosome 9.  


The two underlying pathogenic mechanisms have been proposed for c9orf72 related neurotoxicity. First, the loss of function of c9orf72 caused by G4C2 expansion disrupts mRNA splicing and decreased protein expression resulting in neurotoxicity. But the lack of motor deficit symptoms in mice without c9orf72 expression and non-pathogenic loss of function mutations suggest that the loss-of-function is not sufficient to cause the disease pathology. Second, the gain of toxicity caused by the accumulation of abnormal sense and antisense RNA and the recruitment of RNA binding proteins resulting in the formation of the RNA foci. The RNA foci sequester various RNA binding proteins, dipeptide repeat proteins, TDP-43 protein that hinder cellular function and result in neurotoxicity.

Based on this evidence, the approach to reduce the expression of aberrant mRNA and RNA foci formation is a formidable target for drug discovery. The gene silencing approach is promising but it comes with challenges that including
a) accessibility to the target tissue,
b) requirement of repeated administration and
c) toxicity due to large bolus injection.
Theoretically, a single injection of AAV packaged with a microRNA gene may allow sustained efficacious concentration in the target tissues with a single injection. Indeed, UniQure is developing gene therapy and result from their studies reveal a feasible expression of miRNA in brain tissue, decreased RNA foci in cytoplasm and nuclei cell and non-clinical disease model.

The studies highlight the following key points

a) Characterization of c9orf72 RNA transcripts from normal and disease populations shows a higher level of the first intronic region in ALS patients with variable sequences in each patient. Exonic regions are conserved in both ALS patients and healthy population. 

b) The design and optimization of candidate miRNAs result in specific binding to the intronic and conserved exonic regions of c9orf72 mRNA. 

c) Validation of c90rf72 gene silencing and AAV5 mediated gene delivery in HEK cells and iPSC neurons show successful transduction and expression of microRNA and decrease in c9orf72 RNA and RNA foci


 d) Proof-of-concept of AVV5 mediated gene delivery shows transduction of AAV5 vector on various brain tissue, expression of microRNA transgene and decreased nuclear and cytoplasmic RNA foci in c9orf72_3 transgenic mice.


Figure 1: Decreased RNA foci after treatment with microRNA specific for c90rf72. (Martier et al)

Figure 2: Expression of  GFP proteins in brain tissues after administration of AVV5 via striatum (Martier et al)

Limitation
These proof of concept studies are performed in cell culture model and transgenic mice. Although the mouse model possesses some disease features such as RNA foci formation, the typical motor neuron-related inactivity and neurodegeneration are not observed. Therefore, it is still a pending question on whether this candidate miRNA mediated suppression is sufficient to prevent neurotoxicity and progressive neurodegeneration.

Path forward and challenges
a) The non-clinical and clinical studies to explore safety and tolerability of AAV 5 mediated gene delivery of microRNA.
b) Exploring the immunogenicity against the microRNA and the viral vectors with the administration of the miRNA package viral genome in the striatum.
c) Optimal efficacious dosage/copy of the vector genome with minimal tolerable toxicity.
d) The durability of the transgene expression in non-human primates and clinical subjects

References
AVV5 mediated microRNA delivery for treatment of ALS AVV5 mediated microRNA delivery for treatment of ALS Reviewed by Biotechnology on February 25, 2019 Rating: 5

Immunology: T cell Development & Activation MCQ

February 24, 2019
Multiple Choice Question on T cell Development, Differentiation and Activation

1) T cells (T lymphocytes) are crucial in recognition of antigens presented by self-MHC. The T cell progenitors undergo proliferation and differentiation in thymus and form a mature T cell. Which of the following is the origin for T cell progenitors?
a) Thymus
b) Hepatocytes
c) Bone marrow
d) None of the above

2) What is the characteristic feature of progenitor T cells that have migrated to the thymus?
a) They express T cell receptor/CD3 complex
b) They express CD28 cell adhesion molecule
c) They express CD4 or CD8 co-receptor
d) None of the above

3) Which of the following cell adhesion molecule is present in the T cell progenitors is required for homing of these cells into thymus?
a)CD25
b) CD44
c) IL-2
d) CTLA-4

4) Pre- T cell receptor complex consists of the β chain of TCR & CD3 molecules that are formed during the proliferation of T cells. The function of the pre-TCR complex include except
a) signal productive rearrangement of TCR β for further proliferation
b) suppress further rearrangement of β chain (allelic exclusion)
c) prepare cells for rearrangement of α chain
d) commit T cells for CD4 or CD8 positive T cells

5) Which of the following is the process for T cell development and maturation?
a) Rearrangement of T cell receptor and expression of coreceptors
b) Positive selection of thymocytes bearing receptors that are capable of binding to self-MHC molecules (MHC restriction)
c) Negative selection ensure the affinity receptor self MHC or MHC antigen complex is eliminated (self-tolerance)
d) All of the above

6) During the differentiation of T lymphocytes, the double positive cells are directed to become CD4 + T cell & CD8+ T cell which are MHC II & MHC I restricted respectively. Which of the following process may be correct for T cell differentiation?
a) Intrinsic model- multiple interactions of MHC with double positive (CD4+ & CD8+) instruct the cell to differentiate
b) Stochastic model- CD4 or CD8 expression in switched of randomly
c) Both A and B
d) None of the above

7) The activation of T cell requires the interaction of MHC/peptide of TCR/CD3 complex activation require expression of
a) Transcription factors such as c-Fos, c-Myc, c-Jun etc.
b) Interleukins such as IL-2, IL-3, and IL-6
c) Adhesion molecules such as CD28, CTLA-4
d) All of the above

8) Which of the following cytoplasmic tail of CD4 or CD8 coreceptors are required for phosphorylation of ITAM present of CD3 molecules?
a) Lck
b) ZAP70
c) LAD
d) None of the above

9) The cell surface proteins on T cell bind to B7 on the antigen presenting cells and serve as a secondary signal. Which of the following is true regarding the secondary signal?
a) CD28 is a protein that binds to B7 on APC that acts as costimulatory signals for T cell activation
b) CTLA-4 is a protein that binds to B7 on APC that acts as a suppressor signal for T cell activation
c) Both
d) None

10) Which of the cytokines function in an autocrine manner and induces T cell proliferation after engagement of TCR with antigens interaction and presence of secondary signal?
a) IL-1
b) IL-2
c) IL-3
d) IL-4

11) Cytokines such as IL-2, IFN-γ, TNF-β, play an important role in cell-mediated cytotoxicity and delay hypersensitivity. Which of the following subset of T helper cells are involved in the process
a) T helper 1 CD4+
b) T helper 2 CD4+
c) T helper 1 CD8+
d) T helper 2 CD8+

12) Cytokines such as IL-4, IL-5, IL-6 & IL-10 play an important role in B cell activation and humoral immune response
a) T helper 1 CD4+
b) T helper 2 CD4+
c) T helper 1 CD8+
d) T helper 2 CD8+

13) The regulatory or suppressor T cells express the specific cell surface marker for its function. It is
a) CD4+ CD25+
b) CD4+ CD25-
c) CD4 - CD8 - CD25 +
d) CD4 + CD8 + CD25 +

14) The activated T cells undergo activation-induced cell death (AICD). Which of the following is the effector molecule that is a response for AICD
a) IL-2
b) Fas/Fas ligand
c) IL-4
d) INF-γ

15) T-cell receptor engagement with antigenic peptide MHC may induce T cell activation or the clonal anergy. Which of the following interaction favors T cell activation
a) Fas/Fas ligand
b) B-7 & CTLA-4 interaction
c) B-7 & CD28 interaction
d) B-7 & CD 8 interaction

Multiple Choice Answers
1-c) Bone marrow
2-d) None of the above
3-b) CD44
4-d) commit T cells for CD4 or CD8 positive T cells
5-d) All of the above
6-c) Both A and B
7-d) All of the above
8-a) Lck
9-c) Both
10-b) IL-2
11- a) T helper 1 CD4+
12-b) T helper 2 CD4+
13- a) CD4+ CD25+
14-b) Fas/Fas ligand
15-b) B-7 & CTLA-4 interaction
Immunology: T cell Development & Activation MCQ Immunology: T cell Development & Activation MCQ Reviewed by Biotechnology on February 24, 2019 Rating: 5

Medical Microbiology: Treponema Pallidum & Other Spirochetes MCQ

February 22, 2019
Multiple Choice Question on Treponema Pallidum & Other Spirochetes

1) Which of the following is not the general characteristics of spirochetes?
a) They are gram-negative helical bacteria
b) They are motile and have periplasmic flagella (endoflagella)
c) They reproduce by transverse binary fusion
d) They are obligate aerobes

2) Which of the following subspecies of Treponema pallidum causes endemic syphilis?
a) Treponema carateum
b) Treponema endemicum
c) Treponema pertenue
d) None of the above

3) The RPR (Rapid Plasma Reagin) is a nontreponemal test method used as a screening test for syphilis, which of the following statements is correct about the test?
a) It is an expensive method
b) It often shows a positive result with primary syphilis
c) It often shows a false positive result for syphilis
d) It is a specific test for Treponema pallidum


4) A 30 years old man infected with syphilis develops a skin lesion on his prepuce, the lesion is oval in shape and is painless. What is the specific name for this type of lesions?
a) Nodules
b) Papule
c) Eczema
d) Hard chancre

5) Epidemic Relapsing fever in humans is caused by the infected body louse which acts as a vector and is transmitted to humans, which of the following is the most likely spirochetes?
a) Borrelia recurrentis
b) Leptospira interrogans
c) Borrelia burgdorferi
d) Borrelia hermsii

6) These bacteria are motile, found worldwide and infect kidneys and lever which can lead to infectious jaundice, the bacterial serovars are transmitted to humans through contact with animals and its excreta. What is the likely pathogen?
a) Borrelia burgdorferi
b) Bordetella pertusis
c) Leptospira interrogans
d) Enterococcus faecalis

7) Which of the following statements is most correct about the dark field microscopy for the diagnosis of spirochetes?
a) It is used to observe and detect thin spirochetes suspended in a liquid
b) The specimen is stained and detected under the compound microscope
c) The color of the spirochetes appears grey when observed under dark background
d) It is used to detect the spirochetes from the oral cavity, cerebrospinal fluid

8) Which of the following organisms is NOT the normal flora of the oral cavity?
a) Streptococcus mutans
b) Borrelia buccalis
c) Spirillum minor
d) Candida albicans

9) What stain is mostly used for the detection of Borrelia spp which causes relapsing fever?
a) Gram stain
b) Ziehl-Neelsen stain
c) Giemsa stain
d) Methylene blue

10) The bacteria causing Leptospirosis are difficult to isolate from urine, blood or CSF specimens by culture method, therefore a confirmation test should be done. Which of the following statement is most correct in the diagnosis of the disease?
a) RPR tests for the detection of serogroup antigens from the acute or convalescent phase
b) Antibodies are tested against the serogroup antigens from the acute or convalescent phase
c) Darkfield microscopy method is done to detect the pathogen from serum samples
d) All of the above



11) The diagnosis of Lyme disease is done by two serological tests after the screening test shows positive result thus first confirmatory test is the ELISA, what is the second?
a) Western blotting assay
b) PCR
c) Immunofluorescent assay
d) Southern blotting assay

12) Which of the following spirochetes causes skin disease primarily to individuals who have dark skin color and is transmitted by direct contact or vectors such as fly?
a) Borrelia hermsii
b) Spirillum minor
c) Treponema pallidum subspecies pertenue
d) Treponema carateum

13) Which of the following pathogens cause sexually transmitted diseases?
a) Treponema pallidum subspecies endemicum
b) Treponema pallidum subspecies pallidum
c) Borrelia recurrentis
d) Borrelia hermsii

14) Which of the following statements is not correct about congenital syphilis?
a) The spirochetes are transferred from mother to fetus across the placenta
b) The early manifestations occur in children during the birth
c) Five to ten percentages of children develop cutaneous lesions
d) The infection can lead to stillbirth

15) What is the most likely pathogen related to rat-bite fever?
a) Leptospira interrogans
b) Borrelia hermsii
c) Treponema pallidum subspecies endemicum
d) Spirillum minor


Multiple Choice Answers Review
1-d) They are obligate aerobes
2-b) Treponema endemicum
3-c) It often shows a false positive result for syphilis
4-d) Hard chancre
5-a) Borrelia recurrentis
6-a) Borrelia burgdorferi
7-a) It is used to observe and detect thin spirochetes suspended in liquid
8-c) Spirillum minor
9-c) Giemsa stain
10)- b) Antibodies are tested against the serogroup antigens from acute or convalescent phase
11-a) Western blotting assay
12-d) Treponema carateum
13-b) Treponema pallidum subspecies pallidum
14-c) Five to ten percentages of children develop cutaneous lesions
15-d) Spirillum minor

Medical Microbiology: Treponema Pallidum & Other Spirochetes MCQ Medical Microbiology: Treponema Pallidum & Other Spirochetes MCQ Reviewed by Biotechnology on February 22, 2019 Rating: 5

Immunoinhibition method for Screening Cut point of immunogencity assay with pre-existing antibodies

February 18, 2019
Anti-drug antibody (ADA) assay is a semi-quantitative bioanalytical method that detects the antibodies specific against the drug. The ADA assay utilizes the assay cut point above which the samples are considered positive. The assay cut points are established by analyzing a set of approximately 50 drug-naive individuals with an assumption that these selected samples do not contain any pre-existing antibodies.

While this is true for most ADA assays, in some instances, there is a high probability of prevalent pre-existing antibodies when a closely related antigen is exposed prior to drug administration. The pre-exposure of antigens such as adeno associated viruses (AAVs), Cas9 protein, PEG molecule account for preexisting antibodies in clinical subjects. When the preexisting antibody is prevalent in the study population, the cut point should be established carefully to enable the detection of the clinically significant ADA. Applying the recommended statistical cut point calculation method (Shankar et al, 2008) in these preexisting ADA populations may result in elevated cut points and a higher false-negative rate. To overcome this challenge, the immune-inhibition method is reported and acceptable to the FDA regulatory agencies.

Immunoinhibition method for preexisting antibodies
In the immune-inhibition method, the excess drug is added to the treatment of naïve samples and the cut point is established using these drug-spiked samples. The method is based on the principle that the excess drug inhibits the availability of preexisting antibodies to bind to the capture/detection system. The competitive inhibition by presence excess drug decreases the assay signal of reactive drug-naive samples. The statistical analysis (target 5% false positive rate) of the immunoinhibition method results in a lower cut point compared to the normal cut-point method. It also improves the detection of clinically significant ADA in clinical studies.





Figure 2: Mechanism of Immuno-inhibition: The spiking of free drug to the pre-existing antibody sample result in the formation of the antibody-drug complex and reduce the availability of antibody binding to capture and detection system.

The critical steps for the immune-inhibition method include:
a) Optimization of minimal drug concentration that results in the highest immunoinhibition
b) Spiking drug to each sample and analyze the drug-spiked sample set
c) Derive cut point using the recommended statistical method
d) Determine the false positive rate of the method
a) Optimization of minimal drug concentration that results in the highest immunoinhibition

The first step of the immunoinhibition method is to define the concentration of the drug required to attain maximal inhibitions without any effect on negative controls. The drug concentration for immunoinhibition is evaluated by spiking the varying amount of drug to positive control and determining the concentration that yields maximal signal inhibition (as shown in figure 3).


Figure 3: Determining optimal drug concentration for the immunoinhibition method (Simhadri et al 2018). In this figure, the anti-SaCas9 (188 ng/mL) and anti-SpCas9 (250 ng/mL) antibodies were pre-incubated with increasing concentrations of SaCas9 (A) and SpCas9 (B) antigen. The highest concentration of antigen used (200 μg/mL) inhibited binding of anti-SaCas9 and anti-SpCas9 antibody

b) Spiking drug to each sample and analyze the drug-spiked sample set
The optimized concentration of a drug is spiked into the drug-naive individual serum and analyzed in the assay. The presence of drugs suppresses the assay signal by binding/saturating preexisting antibodies. As shown in figure 4, individuals without drug spike are randomly dispersed with varying reactivity. In contrast. the same samples with drug spike have homogeneously distributed assay signal. Using this data, the cut point calculated at the 95th percentile from the drug inhibition results in lower cut points and a higher positive rate in the population.
Figure 4: Immunoinhibition Method for Cutpoint determination (Schneider et al. 2018)

c) Derive cut point using the recommended statistical method: Click here for details
d) Determine the false positive rate of the method: Click here for details

Other methods for the cut point calculation includes:
a) Removal of pre-existing antibodies using Protein A/G
b) Titer method

References
Determination of Assay Cut point
Schneider et al. 2016 J Immun Methods
Simhadri et al. 2018 Mol Ther Methods & Clin Dev
Immunoinhibition method for Screening Cut point of immunogencity assay with pre-existing antibodies Immunoinhibition method for Screening Cut point of immunogencity assay with pre-existing antibodies Reviewed by Biotechnology on February 18, 2019 Rating: 5

Pre-existing Immune Responses against Cas9 protein (CRISPR) add complexity to CRISPR based Gene Therapy

February 15, 2019
CRISPR is a gene editing tools that are being explored as a potential gene therapy candidate for gene deletions, corrections etc. The high efficiency and specificity of the CRISPR enable its global use in basic research and drug discovery research. The CRISPR consist of two components a) Cas9, a protein that has a nuclease activity b) Guide RNA that is recognizes the target sequence and enable cleavage of target sequence catalyzed by Cas9. Cas9 is a protein is originally isolated from bacteria Staphylococcus aureus and Streptococcus pyogenes. Because Cas9 is originated from human pathogenic bacteria, the prior exposure to the protein may results in pre-existing antibodies and T cell response against Cas9. The pre-existing immunogenicity against the Cas9 pose a risk impacting safety and efficacy of CRISPR mediated gene therapy.

Recent studies have reported that the prevalence of the pre-existing antibodies against Cas9 proteins. Simhari et al. 2018 reported the prevalence of anti-Sa Cas9 & anti-Sp Cas9 antibody to be 10.0% and 2.5% respectively using ELISA assay. Another study by Charlesworth et al. 2018 reported higher prevalence of antibodies using immunoblot assay. Although the small sample size, the co-incidence of T-cell response with the pre-existing antibodies are approximately forty six percent (Charlesworth et al. 2018). The different assay methods were used for determining anti-Cas9 antibodies in these studies, and therefore, the outcome should not be compared. Nevertheless, these studies provided the insight on the pre-existing antibody and T cell response against Cas9 protein.

The Cas9 RNA is generally package into the nanoparticle and delivered into the target cells. Alternatively, Cas9 DNA are also packaged into the viral vectors, delivered into the target tissue and expressed endogenously. But the Cas9 protein are rarely administered directly and it may not directly comes in contact with Cas9 antibodies. Therefore, the pre-existing Cas9 antibodies may have lesser impact on drug efficacy and safety. In contrast, the anti-Cas9 IgG antibody and Cas9 activated interferon gamma release (in vitro) suggest a positive of Cas9 specific T-cell and B-cell responses. And the pre-existing Cas9 specific T-cell activity against Cas9 pose a risk of recognizing the MHC- Cas9 transgene complex thereby activating the cytotoxic T lymphocytes and target cell lysis.

Finally these studies have provided the preliminary data on immunogenicity and substantiated the importance of immunogenicity assessment program for drug development that include
a) Characterize pre-existing antidrug antibodies
b) Identify T cell epitopes, HLA restriction
b) Characterization of T cell responses
c) Clinical consequence of pre-existing anti-Cas9 antibodies and T cell responses in safety and efficacy.

References
Charesworth et al. 2018 bioRxiv
Simhadri et al. 2018 Molecular Therapy: Methods & Clinical Development
Pre-existing Immune Responses against Cas9 protein (CRISPR) add complexity to CRISPR based Gene Therapy Pre-existing Immune Responses against Cas9 protein (CRISPR) add complexity to CRISPR based Gene Therapy Reviewed by Biotechnology on February 15, 2019 Rating: 5

How to develop a reliable, sensitive and specific ADA assays for immunogenicity testing?

February 15, 2019
FDA guidance for Immunogenicity Testing Assays: Development & Validation of Anti-drug Antibody Assays

How to develop reliable, sensitive and specific ADA assays for immunogenicity testing?
The reliable, sensitive and specific ADA assays are crucial for facilitating the understanding of immunogenicity, pharmacokinetics, pharmacodynamics, safety, and efficacy of therapeutic protein products. During development, evaluation of key aspects of the assay such as assay parameters such as sensitivity, specificity, drug tolerance & critical reagent selection and ensure the robust, reproducible and sensitive detection of anti-drug antibodies.

Table 2: Summary of the key assay parameters for developing and validating ADA assays

Assay Parameters
Description
Assessment Method
Cut point
-The level of response of the assay that defines the sample response as positive and negative.
-Screening, confirmation and titer cut points are used for three tiers of the assay.
- Cut-points are critical to minimize the risk of false negative 
- Drug naïve individuals from the target population should be used to derive cut point, if feasible.
- Normal healthy individuals obtained from commercial vendors during development and confirmation by study samples when it becomes available.
- Statistical method for deriving cut points is the preferred method.

Sensitivity and Surrogate Positive Controls

-The lowest concentration at which the antibody preparation consistently produces either positive results.
- TAb, IgG and IgM less than 100 ng/ml.
- IgE assay is less than 10ng/ml.  
- Positive control- Affinity purified antibodies should be used.
- Assay sensitivity may be assessed by testing serial dilution of positive control antibody of known concentration, using individual or pooled matrix from treatment.
- Interpolate values may be used.
Drug Tolerance
The assessment of the sensitivity of the assay in the presence of the expected level of interfering therapeutic protein product.
- Drug tolerance may be assessed by deliberately adding different quantities of the therapeutic protein product
Specificity
The ability of a method to exclusively detect the target analyte. Lack of the specificity leads to false-positive results, obscuring the ADA –PK/PD relationship.
-Drug tolerance may be assessed by deliberately adding closely related proteins to ADAs, including isotype controls.
Selectivity
The ability of the assay to identify ADAs specific to the therapeutic protein product in the presence of other components in the samples
-Matrix interference and minimum required dilution should be assessed by spiking the positive controls in drug naïve sample.
-Should be assessed using drug naïve samples from clinical subjects
Robustness
Assay Robustness is an ability to remain unaffected with a small variation on the temperature, incubations times, buffer characteristics, freeze-thaw cycle etc.
Robustness may be assessed by deliberately introducing the small variation of method and instrument performance
Precision
Precision is a measure of the variability in a series of measurement for the same material run in a method.
Assessed by evaluating the imprecision (%CV) of low, intermediate and high concentration quality controls (HQC, Intermediate QC, and LQC)
Critical Reagent Stability and lot-lot variation
Critical reagents such as capture and detection reagents, positive control antibodies, cell lines, and passages  
Assessed by the ability to remain unchanged with tested storage conditions and durations, lot-to-lot variations, etc.


Figure 1: Isotype Characterization Assay 
Summary
- The reliable, sensitivity, specific and valid ADA assay method development and validation require testing of key aspects of the assay such as cut point, drug tolerance, etc.
- FDA also expects the sponsor to conduct a systematic and methodological assay development and ensure that the assay has sufficient sensitivity to detect ADA that could mediate biological or physiological consequences.
- FDA expects the submission of the validation report with the biologic license application.
-Finally, the current document also provides the recommendation specific for neutralizing antibody assays, selection of assay format, positive controls, sample testing and documentation requirement that are/will be discussed in another subsequent post.

References
Immunogenicity Assessment for Therapeutic Protein Products: US FDA 2014
Guideline on Immunogenicity assessment of therapeutic proteins: EMA 2017
Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products
How to develop a reliable, sensitive and specific ADA assays for immunogenicity testing? How to develop a reliable, sensitive and specific ADA assays for immunogenicity testing? Reviewed by Biotechnology on February 15, 2019 Rating: 5

Medical Microbiology: Haemophilus & Bordetella MCQ

February 11, 2019
Multiple Choice Question on Haemophilus & Bordetella

1) Which of these small, gram-negative microorganisms are important human pathogens?
a) Haemophilus influenzae
b) Bordetella pertussis
c) Haemophilus parainfluenzae
d) A, B and C

2) …..................was one of the leading bacterial pathogens that caused severe upper respiratory tract infections and even death in children during the nineteenth century, it is now rare in countries where routine vaccination is given to children.
a) Haemophilus influenzae
b) Bordetella pertussis
c) Haemophilus influenzae type b
d) Francisella tularensis

3) Which of the following Haemophilus spp require both the X (hemin) and V (NAD) factors for its growth?
a) H. parainfluenzae
b) H. ducreyi
c) H. aprophilus and H. paraaprophilus
d) H. haemolyticus

4) Which of the following is NOT the virulence factors responsible for the pathogenicity of Bordetella pertussis, a gram-negative coccobacillus that causes “whooping cough”?
a) An Endotoxin
b) A fimbriae and hemagglutinin
c) A pertussis toxin
d) A tracheal cytotoxin

5) Which of the following bacteria is responsible for “Malta fever” in humans which is caused primarily by contact with animals or animal products?
a) Haemophilus ducreyi
b) Francisella tularensis
c) Brucella spp
d) Haemophilus parainfluenzae

6) Name the specific food when consumed by humans that can cause “malta fever”?
a) Unpasteurized milk or cheese
b) Uncooked cauliflower
c) Plums
d) Cranberry juice

7) …....................is aerobic, non-spore forming, coco bacillus and can be transmitted to humans through water, soil, animals, also through aerosols
a) Haemophilus influenzae
b) Brucella spp
c) Francisella tularensis
d) Neisseria gonorrohoea

8) Which of the following subspecies of Francisella tularensis is the most virulent for humans?
a) novicida
b) tularensis
c) holartica
d) mediasiatica

9) All of the following are the symptoms caused by the pathogenic Brucella spp, EXCEPT
a) Fever
b) Swollen lymph nodes
c) Abdominal and muscular pain
d) Lesions on eyelids

10) Which of the following is not the general characteristics of Haemophilus ducreyi?
a) Gram-negative coccobacillus
b) It grows well on standard chocolate agar
c) It requires X factor but not the V factor
d) It is susceptible to erythromycin

11) Name the antibiotics group which is commonly NOT used for the treatment of influenza caused by Haemophilus influenzae?
a) Third generation cephalosporins
b) Aminopenicillins with a beta-lactamase inhibitor
c) Chloramphenicol with ampicillin
d) Amoxicillin with penicillin

12) What is a Quellung reaction?
a) The capsule detection test
b) The serological antibody detection test
c) Direct antigen detection test
d) The bacterial motility test

13) The preventive measure for Bordetella pertussis infection is vaccination method, the pertussis vaccine is usually administered in combination with toxoids of Diphtheria and tetanus (DTaP). The pertussis vaccine is primarily important for children, preteens, pregnant women and adults who have never received it, what doses of this vaccine is recommended for children under six years?
a) Six doses of vaccine
b) Three doses of vaccine
c) Five doses of vaccine
d) Four doses of vaccine

14) .................... is the causative agent of the chancroid, one of the most common sexually transmitted disease and is characterized by painful lesions and genital ulcers?
a) Neisseria gonorrohoeae
b) Haemophilus haemolyticus
c) Haemophilus ducreyi
d) Treponema pallidum

15) Which of the following do not prove to be helpful for the treatment of whooping cough?
a) Cough syrups and expectorants
b) DPT vaccine
c) Macrolides
d) None of the above

Multiple Choice Answers
1-d) A, B and C
2-c) Haemophilus influenzae type b
3-d) H. haemolyticus
4-a) An Endotoxin
5-c) Brucella spp
6-a) Unpasteurized milk or cheese
7-c) Francisella tularensis
8-b) tularensis
9-d) Lesions on eyelids
10-b) It grows well on standard chocolate agar
11-d) Amoxicillin with penicillin
12-a) The capsule detection test
13-c) Five doses of vaccine
14-c) Haemophilus ducreyi
15-a) Cough syrups and expectorants

Medical Microbiology: Haemophilus & Bordetella MCQ Medical Microbiology: Haemophilus & Bordetella MCQ Reviewed by Biotechnology on February 11, 2019 Rating: 5

Medical Microbiology: Mycobacteria MCQ

February 06, 2019
Multiple Choice Question on Mycobacteria tuberculosis and Mycobacteria leprae

1) Mycobacteria are acid-fast positive bacteria because of:
a) the presence of lipopolysaccharide in the bacterial cell wall
b) the presence of mycolic acid in the bacterial cell wall
c) the presence of lipids
d) Both B and C options given above

2) Which of the following first-line antibiotics are usually resistant to Mycobacterium tuberculosis?
a) Isoniazid and ciprofloxacin
b) Isoniazid and Rifampin
c) Rifampin and ciprofloxacin
d) Rifampin and streptomycin

3) What is the mechanism responsible for antibiotic resistance in Mycobacterium tuberculosis?
a) Mutations in DNA gyrase gene
b) Alterations in beta-lactamase
c) Mutations in the catalase-peroxidase gene
d) Alterations in RNA polymerase


4) All of the following are the examples of selective media used for culture of Mycobacterium tuberculosis, except?
a) Inspissated egg media
b) Middlebrook 7H10/7H11 media with antibiotics
c) Dubos media
d) Middlebrook 7H10 media without antibiotics

5) Which of the following statement is true about the tuberculin test and purified protein derivative (PPD)?
a) The presence of intradermal skin induration is observed in 6 to 8 hours after being applied
b) The redness of skin or erythema is also measured while reading the tuberculin test
c) A positive tuberculin test means that a person was infected with M. tuberculosis in the past and continues to carry the viable organism
d) A positive PPD test indicates that a person can never be infected with M. tuberculosis

6) Which of the following is fast growing, non-chromogenic rod bacilli?
a) Mycobacterium ulcerans
b) Mycobacterium avium complex
c) Mycobacterium leprae
d) Mycobacterium fortuitum

7) All of the following are the symptoms of pulmonary tuberculosis, EXCEPT?
a) Weakness and fatigue
b) Decreased body temperature
c) Weight loss
d) Severe prolonged cough with sputum or blood

8) Which of these following categories of people do not require the test for TB infection?
a) A 30 years old man traveling from India to the USA for the first time
b) A 45 years old woman contradicted with HIV infection
c) A 7 years old healthy girl living together with her grandmother who has tuberculosis infection
d) None of the above

9) Which of the following bacteria causes lung infection and is the most common non-tuberculous mycobacterial infection associated with AIDS patients?
a) Mycobacterium avium complex
b) Mycobacterium leprae
c) Mycobacterium gordonae
d) Mycobacterium gastri

10) A 40 years old patient who has clinical symptoms pulmonary tuberculosis for the past two months. The chest x-ray examination revealed a typical feature of tuberculosis infection. A photochromogenic (orange pigment when exposed to UV light) acid-fast rod bacterium was isolated from the sputum sample. The identified bacteria is:
a) Mycobacterium avium
b) Mycobacterium tuberculosis
c) Mycobacterium kansasii
d) Mycobacterium leprae


11) All of the given are the distinguishing characteristics of Mycobacterium leprae, EXCEPT:
a) Acid-fast bacilli
b) cannot be isolated by in-vitro culture method
c) is a human and as well as animal pathogen
d) can be isolated by in-vivo culture method

12) Which one of the following acid-fast rod bacilli can take up to ten years for its growth in host cells and causes skin infections?
a) Mycobacterium tuberculosis
b) Mycobacterium avium complex
c) Mycobacterium leprae
d) Nocardia spp

13) Other than Mycobacterium tuberculosis which of the given bacteria causes tuberculosis infection in animals and can be transmitted to human by consumption of milk and other animal products?
a) Mycobacterium ulcerans
b) Mycobacterium leprae
c) Mycobacterium bovis
d) Mycobacterium abscessus

14) The treatment regimen for initial therapy of tuberculosis caused by M. tuberculosis includes?
a) Streptomycin and rifampin
b) Isoniazid, streptomycin, and ethambutol
c) Rifampin, isoniazid, and ciprofloxacin
d) Isoniazid, rifampin, pyrazinamide, and ethambutol

15) What is the interferon-gamma release assay?
a) The antigen detection test for the Mycobacterium spp
b) The DNA detection test in Mycobacterium spp
c) The test used as an alternative tuberculin skin test in latent tuberculosis
d) The test used as an alternative tuberculin skin test in active tuberculosis


Multiple Choice Answers Review
1-d) Both B and C options given above
2-b) Isoniazid and Rifampin
3-c) Mutations in the catalase-peroxidase gene
4-d) Middlebrook 7H10 media without antibiotics
5-c) A positive tuberculin test means that a person was infected with M. tuberculosis in the past and continues to carry the viable organism
6- d) Mycobacterium fortuitum
7- b) Decreased body temperature
8- d) None of the above 9-a) Mycobacterium avium
10-c) Mycobacterium kansasi
11-c) is a human and as well as animal pathogen
12-c) Mycobacterium leprae
13-c) Mycobacterium bovis
14-d) Isoniazid, rifampin, pyrazinamide, and ethambutol
15-a) The antigen detection test for the Mycobacterium spp
Medical Microbiology: Mycobacteria MCQ Medical Microbiology: Mycobacteria MCQ Reviewed by Biotechnology on February 06, 2019 Rating: 5

Immunology of T Cell Receptors (TCR gene structure and T cell Functions): MCQ

February 02, 2019
Multiple Choice Question of T-cell Receptors
1) T cells express a transmembrane protein that recognizes the peptide-loaded MHC (pMHC) to activate T cell-mediated immune response. The T-cell receptor is a
a) protein of immunoglobulin superfamily
b) seven transmembrane G protein family
c) tyrosine kinase receptor superfamily
d) None of the above

2) T cell receptor is a heterodimer consist of
a) alpha and beta chain
b) gamma and delta chain
c) Both of the above
d) None of the above

3) Which of the following statement is FALSE regarding the T-cell receptor
a) The alpha and beta chains of TCR are proteins of the immunoglobulin superfamily
b) Both alpha and beta chains consist of one variable domain and one constant domain
c) The TCR resemble Fab fragment of an immunoglobulin
d) The variable region is present in the C terminal region of the TCR

4) Which of the following is NOT the characteristic feature of T cell receptor
a) T-cell lineage Exclusivity
b) Affinity Maturation
c) The combinatorial joining of VDJ
d) Somatic hypermutation
5) Gamma/delta T cell receptors are present in a subset of T-cells and play role in unconventional recognition of antigens. All of the following statements are correct EXCEPT
a) The gamma/delta T-cell receptors are co-expressed with alpha/beta T-cell receptors
b) The gamma/delta T cell receptors undergo allelic exclusion when alpha/beta are expressed and vice versa
c) The gamma/delta T cell receptors recognize the phospholipid antigens
d) All of the above

6) Which of the following statement is true regarding the TCR gene organization and rearrangement?
a) The alpha & gamma genes consist of multiple variable (V), joining (J) and one constant gene
b) The beta & delta genes consist of multiple variable (V), diversity (D), joining (J) genes and one constant gene.
c) Both of the above
d) None of the above

7) Which of the following process contribute to the diversity of TCR?
a) Somatic hypermutation
b) Junctional flexibility
c) Genetic recombination
d) None of the above
8) Once the TCR binds to the pMHC, the signal transduction mediates the T cell activation via CD3 molecules. The CD3 molecules consist of
a) Delta & Epsilon heterodimer
b) Gamma and Epsilon heterodimer
c) Zeta homodimer
d) All of the above
9) The alpha/beta chain of TCR has three complementarity determinant regions CDR1, CDR2, and CDR3. Which of the following CDR binds to the antigen peptide?
a) CDR1
b) CDR2
c) CDR3
d) All of the above

10) Which of the following is NOT the function of the T-cell receptor
a) Antigen Recognition
b) Facilitate binding of co-receptor
c) Serve as the signal transducer
d) Induce signal transduction via a CD3 protein complex
11) Which of the following CD3 molecule is required for assembly of TCR-CD3 complex into the plasma membrane?
a) Gamma
b) Delta
c) Epsilon
d) Zeta

12) Which of the following protein of TCR/CD3 complex do not consist of ITAM domain required for signal transduction?
a) alpha/ beta chain of TCR
b) gamma/epsilon heterodimer of the CD3 complex
c) delta/epsilon heterodimer of the CD3 complex
d) Zeta homodimer of the CD3 complex

13) Which of the following subunits have three ITAM domains?
a) Gamma
b) Delta
c) Epsilon
d) Zeta

14) In addition to the TCR-CD3 complex, the T cell activation requires the engagement of co-receptors with the MHC of the antigen presenting cells. Which of the following co-receptor binds to MHC-I APC
a) CD4
b) CD8
c) CD20
d) None of the above

15) In addition to the TCR-CD3 complex, the T cell activation requires the engagement of co-receptors with the MHC of the antigen presenting cells. Which of the following co-receptor binds to MHC-II APC
a) CD4
b) CD8
c) CD20
d) None of the above

Multiple Choice Answers Review
1-a) protein of immunoglobulin superfamily
2-c) Both of the above
3-d) The variable region is present in the C terminal region of the TCR
4-d) Somatic hypermutation
5-a) The gamma/delta T-cell receptors are co-expressed with alpha/beta T-cell receptors
6-c) Both of the above
7-b) Junctional flexibility
8-d) All of the above
9-c) CDR3
10-c) Serve as the signal transducer
11-d) Zeta
12- a) alpha/ beta chain of TCR
13-d) Zeta
14-b) CD8
15-a) CD4
Immunology of T Cell Receptors (TCR gene structure and T cell Functions): MCQ Immunology of  T Cell Receptors (TCR gene structure and T cell Functions): MCQ Reviewed by Biotechnology on February 02, 2019 Rating: 5
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